Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

Background -- N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a synthetic retinoid with potent pro-apoptotic activity against several types of cancer, but little is known regarding mechanisms leading to chemoresistance. Ceramide and, more recently, other sphingolipid species (e.g., dihydroceramide and dihydrosphingosine) have been implicated in 4-HPR-mediated tumor cell death. Because sphingolipid metabolism has been reported to be altered in drug-resistant tumor cells, we studied the implication of sphingolipids in acquired resistance to 4-HPR based on an acute lymphoblastic leukemia model. Methods -- CCRF-CEM cell lines resistant to 4-HPR were obtained by gradual selection. Endogenous sphingolipid profiles and in situ enzymatic activities were determined by LC/MS, and resistance to 4-HPR or to alternative treatments was measured using the XTT viability assay and annexin V-FITC/propidium iodide labeling. Results -- No major crossresistance was observed against other antitumoral compounds (i.e. paclitaxel, cisplatin, doxorubicin hydrochloride) or agents (i.e. ultra violet C, hydrogen peroxide) also described as sphingolipid modulators. CCRF-CEM cell lines resistant to 4-HPR exhibited a distinctive endogenous sphingolipid profile that correlated with inhibition of dihydroceramide desaturase. Cells maintained acquired resistance to 4-HPR after the removal of 4-HPR though the sphingolipid profile returned to control levels. On the other hand, combined treatment with sphingosine kinase inhibitors (unnatural (dihydro)sphingosines ((dh)Sph)) and glucosylceramide synthase inhibitor (PPMP) in the presence or absence of 4-HPR increased cellular (dh)Sph (but not ceramide) levels and were highly toxic for both parental and resistant cells. Conclusions -- In the leukemia model, acquired resistance to 4-HPR is selective and persists in the absence of sphingolipid profile alteration. Therapeutically, the data demonstrate that alternative sphingolipid-modulating antitumoral strategies are suitable for both 4-HPR-resistant and sensitive leukemia cells. Thus, whereas sphingolipids may not be critical for maintaining resistance to 4-HPR, manipulation of cytotoxic sphingolipids should be considered a viable approach for overcoming resistance.

Regulation of Energy Metabolism by the Extracytoplasmic Function (ECF) σ Factors of Arcobacter butzleri

11 p.

Multi-drug resistance profiles and the genetic features of Acinetobacter baumannii isolates from Bolivia

Introduction: Acinetobacter baumannii is opportunistic in debilitated hospitalised patients. Because information from some South American countries was previously lacking, this study examined the emergence of multi-resistant A. baumannii in three hospitals in Cochabamba, Bolivia, from 2008 to 2009. Methodology: Multiplex PCR was used to identify the main resistance genes in 15 multi-resistant A. baumannii isolates. RT-PCR was used to measure gene expression. The genetic environment of these genes was also analysed by PCR amplification and sequencing. Minimum inhibitory concentrations were determined for key antibiotics and some were determined in the presence of an efflux pump inhibitor, 1-(1-napthylmethyl) piperazine. Results: Fourteen strains were found to be multi-resistant. Each strain was found to have the bla(OXA-58) gene with the ISAba3-like element upstream, responsible for over-expression of the latter and subsequent carbapenem resistance. Similarly, ISAba1, upstream of the bla(ADC) gene caused over-expression of the latter and cephalosporin resistance; mutations in the gyrA(Ser83 to Leu) and parC (Ser-80 to Phe) genes were commensurate with fluoroquinolone resistance. In addition, the adeA, adeB efflux genes were over-expressed. All 15 isolates were positive for at least two aminoglycoside resistance genes. Conclusion: This is one of the first reports analyzing the multi-drug resistance profile of A. baumannii strains isolated in Bolivia and shows that the over-expression of thebla(OXA-58), bla(ADC) and efflux genes together with aminoglycoside modifying enzymes and mutations in DNA topoisomerases are responsible for the multi-resistance of the bacteria and the subsequent difficulty in treating infections caused by them.

Changes in white adipose tissue metabolism induced by resveratrol in rats

Background: A remarkable range of biological functions have been ascribed to resveratrol. Recently, this polyphenol has been shown to have body fat lowering effects. The aim of the present study was to assess some of the potential underlying mechanisms of action which take place in adipose tissue. Methods: Sixteen male Sprague-Dawley rats were randomly divided into two groups: control and treated with 30 mg resveratrol/kg body weight/d. All rats were fed an obesogenic diet and after six weeks of treatment white adipose tissues were dissected. Lipoprotein lipase activity was assessed by fluorimetry, acetyl-CoA carboxylase by radiometry, and malic enzyme, glucose-6P-dehydrogenase and fatty acid synthase by spectrophotometry. Gene expression levels of acetyl-CoA carboxylase, fatty acid synthase, lipoprotein lipase, hormone-sensitive lipase, adipose triglyceride lipase, PPAR-gamma, SREBP-1c and perilipin were assessed by Real time RT-PCR. The amount of resveratrol metabolites in adipose tissue was measured by chromatography. Results: There was no difference in the final body weight of the rats; however, adipose tissues were significantly decreased in the resveratrol-treated group. Resveratrol reduced the activity of lipogenic enzymes, as well as that of heparin-releasable lipoprotein lipase. Moreover, a significant reduction was induced by this polyphenol in hormone-sensitive lipase mRNA levels. No significant changes were observed in other genes. Total amount of resveratrol metabolites in adipose tissue was 2.66 +/- 0.55 nmol/g tissue. Conclusions: It can be proposed that the body fat-lowering effect of resveratrol is mediated, at least in part, by a reduction in fatty acid uptake from circulating triacylglycerols and also in de novo lipogenesis.

A Delphi Process to Optimize Quality and Performance of Drug Evaluation in Neonates

Background: Neonatal trials remain difficult to conduct for several reasons: in particular the need for study sites to have an existing infrastructure in place, with trained investigators and validated quality procedures to ensure good clinical, laboratory practices and a respect for high ethical standards. The objective of this work was to identify the major criteria considered necessary for selecting neonatal intensive care units that are able to perform drug evaluations competently. Methodology and Main Findings: This Delphi process was conducted with an international multidisciplinary panel of 25 experts from 13 countries, selected to be part of two committees (a scientific committee and an expert committee), in order to validate criteria required to perform drug evaluation in neonates. Eighty six items were initially selected and classified under 7 headings: "NICUs description - Level of care'' (21), "Ability to perform drug trials: NICU organization and processes (15), "Research Experience'' (12), "Scientific competencies and area of expertise'' (8), "Quality Management'' (16), "Training and educational capacity'' (8) and "Public involvement'' (6). Sixty-one items were retained and headings were rearranged after the first round, 34 were selected after the second round. A third round was required to validate 13 additional items. The final set includes 47 items divided under 5 headings. Conclusion: A set of 47 relevant criteria will help to NICUs that want to implement, conduct or participate in drug trials within a neonatal network identify important issues to be aware of. Summary Points: 1) Neonatal trials remain difficult to conduct for several reasons: in particular the need for study sites to have an existing infrastructure in place, with trained investigators and validated quality procedures to ensure good clinical, laboratory practices and a respect for high ethical standards. 2) The present Delphi study was conducted with an international multidisciplinary panel of 25 experts from 13 countries and aims to identify the major criteria considered necessary for selecting neonatal intensive care units (NICUs) that are able to perform drug evaluations competently. 3) Of the 86 items initially selected and classified under 7 headings - "NICUs description - Level of care'' (21), "Ability to perform drug trials: NICU organization and processes (15), "Research Experience'' (12), "Scientific competencies and area of expertise'' (8), "Quality Management'' (16), "Training and educational capacity'' (8) and "Public involvement'' (6) - 47 items were selected following a three rounds Delphi process. 4) The present consensus will help NICUs to implement, conduct or participate in drug trials within a neonatal network.

Biofunctionalization of alginate microcapsules: advances in cell-based drug delivery systems

172 p.

Modular Multimodal Iron Oxide-Based Nanocarriers for Image-Guided dsRNA Immunostimulation and Platinum Anticancer Drug Design

237 p.

Exploring new labelling strategies for boronated compounds: towards fast development and efficient assessment of BNCT drug candidates

208 p.